A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-robust Proteomics

Bache, Nicolai; Geyer, Philipp E.; Bekker-Jensen, Dorte B.; Hoerning, Ole; Falkenby, Lasse Gaarde; Treit, Peter V.; Doll, Sophia; Paron, Igor; Müller-Reif, Johannes B.; Meier, Florian; Olsen, Jesper V.; Vorm, Ole; Mann, Matthias

doi:10.1074/mcp.tir118.000853

Cited by 315 publications

(360 citation statements)

References 34 publications

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“…Technological advancements in instrumentation, coupling to mass spectrometer and improvements in separation performance have accompanied this development . Even though nanoLC–MS is still the most commonly used analysis platform for advanced proteomics , several studies have demonstrated that combination of nanoLC–MS and CE–MS methods increase the number of identified proteins as they are orthogonal and provide complementary results .…”

Section: Introductionmentioning

confidence: 99%

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

Ramos

González

Sosa‐Acosta

et al. 2019

J of Separation Science

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Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.

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Section: Introductionmentioning

confidence: 99%

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

Ramos

González

Sosa‐Acosta

et al. 2019

J of Separation Science

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“…Recently, the Evosep One system was described and applied to the analysis of offline-fractionated peptides on a Q-Exactive HF-X MS system 9 of MS utilization metric that is the same as the 'injection efficiency' value calculated by the authors of the above study revealed that the Evosep system substantially outperformed the standard Easy nanoLC system (88% vs. 52% injection efficiency). However, examination of MS1 chromatograms and MS2 scan triggering revealed the presence of extended empty elution windows in the early fractions in the set for either system ( Figure S2a-d), as described previously 14 .…”

Section: High-flow Nano-chromatography Columns Facilitate Rapid Analymentioning

confidence: 96%

High-flow Nano-chromatography Columns Facilitate Rapid and High-quality Proteome Analysis on Standard Nano-LC Hardware

Hughes

Adomat

LeBihan

et al. 2018

Preprint

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Optimizing the acquisition of proteomics data collected from a mass spectrometer (MS) requires careful selection of processed material quantities, liquid-chromatography (LC) setup, and data acquisition parameters. The small internal diameter (ID) columns standardly used in nano-chromatography coupled MS result in long per injection overhead times that require sacrifices in design of offline-fractionation and data acquisition schemes. As cohort sizes and the numbers of samples to be analyzed continue to increase, there is a need to investigate methods for improving the efficiency and time of an acquisition (LC + MS). In this work, the ability to improve throughput in single runs or as part of an in-depth proteome analysis of a fractionated sample using standard LC hardware is investigated. Capitalizing on the increased loading capacity of nanochromatography columns with larger IDs, substantially improved throughput with no reduction in detection sensitivity is achieved in single-injection proteomeanalyses. An optimized 150 μm ID column setup is paired with an offline fractionation-concatenation scheme to demonstrate the ability to perform in-depth proteome analysis on-par with current state-of-the-art studies, while minimizing sample loading overhead. Together, these data demonstrate an easy and effective means to improve sample analysis throughput with no reduction in data quality using an approach that is applicable to any standard nano-LC hardware.3

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“…Many improvements to sample throughput are centered on the recent popularization of microflow LC–MS/MS for both targeted and untargeted proteomics. Microflow LC–MS/MS allows for shorter gradients, quicker column washing, and equilibration, as well as better retention time stability and robustness compared to nanoflow LC–MS . Particularly interesting, a novel LC system utilizes automated offline SPE/desalting and preformed gradients at microflow flow rates for extremely robust and high throughput chromatography suitable for clinical applications …”

Section: Role Of Emerging Technologies In Advancing Biomarker Discovementioning

confidence: 99%

Targeted and Untargeted Proteomics Approaches in Biomarker Development

et al. 2020

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An enormous amount of research effort has been devoted to biomarker discovery and validation. With the completion of the human genome, proteomics is now playing an increasing role in this search for new and better biomarkers. Here, what leads to successful biomarker development is reviewed and how these features may be applied in the context of proteomic biomarker research is considered. The "fit-for-purpose" approach to biomarker development suggests that untargeted proteomic approaches may be better suited for early stages of biomarker discovery, while targeted approaches are preferred for validation and implementation. A systematic screening of published biomarker articles using MS-based proteomics reveals that while both targeted and untargeted technologies are used in proteomic biomarker development, most researchers do not combine these approaches. i) The reasons for this discrepancy, (ii) how proteomic technologies can overcome technical challenges that seem to limit their translation into the clinic, and (iii) how MS can improve, complement, or replace existing clinically important assays in the future are discussed.

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A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-robust Proteomics

Cited by 315 publications

References 34 publications

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

High-flow Nano-chromatography Columns Facilitate Rapid and High-quality Proteome Analysis on Standard Nano-LC Hardware

Targeted and Untargeted Proteomics Approaches in Biomarker Development

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